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120 Activity of any ion (with charge z)

2.7*10^-3*Ca^1.07 x PO4^0.70 x (pH - 4.5) ^ 6.8 Equation 12. Ionic Strength = ---

Cit^0.20 x V^1.31

The other important finding is that activity of Ca2+ in the urine of healthy adults was found to be quite variable throughout the day.

121 STONE FORMATION INHIBITORS AND CHELATING AGENTS:

CITRATE:

Citrate is the most important calcium chelator. It binds calcium and inhibits nucleation and growth of calcium crystals. It chelates calcium at 1:1 stoichiometry and when citrate concentration is more than calcium concentration virtually free Ca2+ activity is nil. Hypocitraturia is a significant risk factor for developing calcium stones. By citrate excretion, urinary bases can be excreted without raising urine pH; thus it maintains divalent phosphate concentration low and prevents calcium phosphate precipitation. [43]

Citrate has multiple functions in mammalian urine and the two most important ones are as a chelator for urinary calcium, and as a physiologic urinary base. It is a tricarboxylic acid cycle intermediate and the majority of citrate reabsorbed by the proximal tubule is oxidized to electroneutral end products so H+ is consumed in the process rendering citrate a major urinary base (Figure 49). Calcium associates in a one-to-one stoichiometry. The highest affinity and solubility is a monovalent anionic (Ca2+Citrate3-)- complex. [68]

122 Citrate exists mostly as a tricarboxylate at plasma pH, but in the proximal tubule lumen, because of apical H+ transport, citrate3- is titrated (citrate3-/citrate2- pK 5.7–6.0) and is taken up in protonated form as citrate2-_{. The Km for dicarboxylates ranges between 0.3 μM and 1 μM.}

Transport of one divalent anion substrate is coupled to three Na+ ions. (Figure 50) [68] Equation 13: Citrate3- + H+ Æ Citrate2- pK 5.7– 6.0

Figure 50. Proximal tubule citrate absorption and metabolism. The Na+K+ATPase generates the low cell [Na+]. As a secondary active transporter NaDC1 uses the electrochemical gradient to pick up filtered citrate, which metabolized in the cytoplasm or the mitochondria. Ambient and cytoplasmic pH increase citrate uptake and metabolism. (1) Acidification of urinary lumen titrates citrate to the divalent transported species; (2) NaDC1 is directly activated by pH and chronic low pH increases expression of NaDC1 (circled arrow); (3) Intracellular acidification increases the expression of ATP citrate lyase and aconitase (circled arrows). [68]

C NaD C1 Na+ Na+ Cit2- AT     3 3 K _K 3 3 Cit2- Cit3- H+    TCA l OA2- Acetyl-CoA CO2 H2O

2H

123 Sodium Dicarboxylate Cotransporter 1 (NaDC1) is found on apical membranes of the renal proximal tubule cells where it mediates absorption of tricarboxylic acid cycle intermediates from the glomerular filtrate or the intestinal lumen. The preferred substrates of NaDC1 are 4-carbon dicarboxylates such as succinate, fumarate, and α-ketoglutarate.

Sodium Dicarboxylate Cotransporter 3 (NaDC3) has a wider tissue distribution and much broader substrate specificity than NaDC1. NaDC3 is expressed on basolateral membranes in renal proximal tubule cells, as well as the liver, brain, and placenta. The basolateral location of NaDC3 was mapped to a motif in its amino-terminal cytoplasmic domain. Like NaDC1, NaDC3 is sodium- coupled and electrogenic so it is very unlikely that NaDC3 will mediate citrate efflux from the

proximal tubule into the peritubular space. [68]

Acidic proximal cells favor tubular reabsorption of citrate and thus hypocitraturia, while alkalosis reduces tubular reabsorption and increases urinary citrate excretion. Potassium- magnesium citrate has been used as prophylaxis against recurrent calcium oxalate nephrolithiasis and it has been recommended as a treatment for nephrocalcinosis to increase urinary pH and citrate concentration, but citrate after absorption will be metabolized to bicarbonate by the liver. [65] _Hence

plasma ionized calcium around 1-1.5 mmol/l is needed for many physiologic reactions, plasma concentration of citrate is very low around 0.16 mmole/l. Therefore, the final urinary excretion of citrate is determined by reabsorption in the proximal tubule and the most important regulator of citrate reabsorption is proximal tubule cell pH. Acid loading increases citrate absorption by four mechanisms: (Figure 50)

124 1. Low luminal pH titrates citrate3- to citrate2- (pK of 5.7-6.0) which is the preferred

transported species;

2. NaDC1 is also gated by pH such that low pH acutely stimulates its activity;

3. Intracellular acidosis increases expression of the NaDC1 transporter and insertion of NaDC1 into the apical membrane;

4. Intracellular acidosis stimulates enzymes that metabolize citrate in the cytoplasm and mitochondria. This is a well concerted response and an appropriate response of the proximal tubule to cellular acidification is hypocitraturia. Although perfectly adaptive from an acid-base point of view, this response is the detrimental to the prevention of calcium chelation.

All conditions that lead to proximal tubular cellular acidification (e.g., distal renal tubular acidosis, high-protein diet, potassium deficiency) are clinical risk factors for calcareous nephrolithiasis. Hypocitraturia can cause kidney stones by itself or by acting with other risk factors such as hypercalciuria. Therapy with potassium citrate has been shown to reverse the biochemical defect and reduce stone recurrence. [68,134]

Citrate also increases the activity of some macromolecules in the urine like Tamm-Horsfall protein that inhibit calcium oxalate aggregation further. Citrate seems able to increase the expression of urinary osteopontin as well. [43]

125 TAMM-HORSFALL PROTEIN:

Tamm-Horsfall protein, also known as uromodulin, is expressed by the thick ascending limb of Henle’s epithelial cells. Tamm-Horsfall protein is the most abundant protein found in urine. Humans produce up to 100 mg of Tamm-Horsfall protein daily in urine (1.5umol/d). There is a significant correlation between the concentration of Tamm-Horsfall protein and citrate in stone former patients. Tamm-Horsfall protein plays dual roles in the formation of Calcium oxalate stones. The inhibitory effect of Tamm-Horsfall protein on crystal aggregation and growth has been described both for calcium oxalate and hydroxyapatite stones. In a rat model of nephrolithiasis, Tamm-Horsfall protein was specifically associated with reduced renal crystal deposits. Approximately, 16% of mice deficient for Tamm-Horsfall protein spontaneously developed calcium crystals in the kidneys. Calcium overload in these mice resulted in an aggravation of calcium crystal formation (76% of the Tamm-Horsfall protein mice), whereas wild-type littermates were still without calcium crystals. Interestingly, Osteopontin expression (see below) is induced in Tamm-Horsfall protein exposed to calcium overload, suggesting a synergistic action of both these proteins. In some humans with calcium oxalate nephrolithiasis, a molecular abnormality of THP could be detected. Other studies showed decreased urinary levels of Tamm-Horsfall protein in patients with nephrolithiasis. In a recent analysis, it could be shown that urinary macromolecular inhibition of crystal adhesion to renal epithelial cells was impaired in male stone formers and related to a relative Tamm-Horsfall protein deficiency. [43]

126 Osteopontin:

Osteopontin, also known as uropontin or nephropontin, is a major component of renal stones. An average of 4 mg of Osteopontin is secreted into urine per day. Osteopontin can inhibit nucleation, growth, and aggregation of calcium oxalate crystals in vitro. Interestingly, Osteopontin can increase the adhesion force between a carboxylate tip and a specific crystal surface. Using immunogold labeling, Osteopontin was shown to be localized mainly on the surfaces of the apatite crystal phase at the junction of crystal organic layers. In vitro experiments revealed that Osteopontin concentrations ranging from 16 to 28 nM are sufficient for a 50% reduction in crystal growth rate and aggregation of calcium oxalate monocrystals, respectively. Mean urine Osteopontin concentrations of 131 nM therefore indicate that Osteopontin may also act in vivo. Mice deficient for Osteopontin develop renal calcium oxalate stones when exposed to high levels of oxalate, but not under normal conditions. In that study, hyperoxaluria in wild-type littermates resulted in an up-regulation of renal Osteopontin expression. [43]

To what extent Osteopontin is involved in human nephrolithiasis is less clear, as there are reports of reduced Osteopontin concentration in stone formers, while other researchers could not detect a difference in urinary Osteopontin levels between stone formers and healthy individuals.

127 Glycosaminoglycans:

Chondroitin sulfate, heparan sulfate, and hyaluronic acid are the best studied glycosaminoglycans with respect to nephrolithiasis. Chondroitin sulfate delays nucleation, while dermatan sulfate inhibits nucleation. Hyaluronic acid, which is found on pericellular matrices, is thought to be the key binding substance for crystals at the surfaces of renal tubular cells. In primary cultures of human tubular cells, intact distal tubular epithelium could not bind crystals, while crystal retention by damaged distal tubular epithelium depended on the expression of hyalonuric acid-, CD44-, and osteopontin- rich cell coats. A rat study showed that during the process of nephrolithiasis, there was an increased expression of heparan sulfate in both distal and proximal tubules. In a canine tubular cells study it was found that synthesis of glycosaminoglycans may increase protection from toxic insults of calcium oxalate crystals and oxalate ions. Some studies in humans show that decreased urinary glycosaminoglycan levels are more common in patients with stone formation. However, other studies could not demonstrate a major relationship between urinary glycosaminoglycan excretion and calcium stone formation. [43]

Renal Handling and Diurnal excretion Variation of magnesium:

The average daily dietary intake of magnesium in adults is 12 mmol, with the minimum daily dietary intake of 0.5 to 4 mmol. Of the dietary magnesium intake, 30% to 50% is normally absorbed, but this can increase to 75% on a low magnesium diet, and decrease to 24% on a high magnesium diet. Urinary excretion normally accounts for about 4 mmol of magnesium output per day. [68]

128 Seventy percent to 80% of serum magnesium is freely filtered at the glomerulus, of which most is reabsorbed along the length of the nephron. Only 5-15% of filtered magnesium will be absorbed in the proximal tubule. However, 60-70% of filtered magnesium will be absorbed in the thick ascending limb of Henle and 5-10% of it will be absorbed in the distal collecting tubule and connecting tubule. Only about 3% of filtered magnesium normally appears in the final urine. During severe dietary magnesium deprivation, the kidney avidly retains magnesium. Under these circumstances, urinary magnesium excretion may be reduced to less than 1 mmol/day (and often less than 0.5 mmol/day), and the fractional excretion of filtered magnesium (FeMg) to less than 1%. Magnesium reabsorption is not, in fact, saturable with respect to luminal magnesium concentration in any segment of the nephron, but is inhibited in a concentration-dependent manner by increasing peritubular magnesium levels in the thick ascending limb of Henle, giving rise to an

apparent Tm effect in studies of whole kidney clearance. [68]

Other Factors:

Nephrocalcin belongs to the osteocalcin family, which constitutes up to 1–2% of total bone protein. Nephrocalcin binds strongly to apatite and calcium. It is expressed in the kidney and depends on vitamin K availability for c-carboxylation and thus activation. Nephrocalcin inhibits nucleation of calcium oxalate monohydrate crystals in vitro. Some patients with renal stones produce an abnormal nephrocalcin lacking the c-carboxyglutamic acid and thus failing to inhibit crystallization functionally. Calgranulin, also known as calprotectin, is a member of the calcium binding S100 family. Calgranulin is a potent inhibitor of calcium oxalate crystal growth and aggregation and can be detected within urinary calcium stones.Urinary prothrombin fragment 1 is present in calcium stones as well and is an inhibitor of calcium oxalate crystallization in urine in vitro. During blood coagulation, prothrombin is ultimately degraded to three fragments: thrombin, fragment 1, and fragment 2, respectively. In patients

129

with calcium oxalate calculi, the c-carboxyglutamic acid composition of urinary prothrombin fragment 1 and its ability to inhibit calcium oxalate crystal growth was described to be significantly decreased. Bikunin, the light chain of inter-a-inhibitor, prevents the adhesion of calcium oxalate crystal to renal tubular cells in human urine. Normally, bikunin expression is mostly limited to the proximal tubules; however, hyperoxaluria in rats resulted in an increased expression of bikunin. Urinary bikunin levels were approximately 50% lower in patients who form calcium oxalate stones compared with normal volunteers. Phytate, the principal storage molecule of phosphorous in many plants, can inhibit calcium oxalate crystal formation in vitro. In the Nurses Health Study II, women in the highest quintile of phytate intake had a reduced relative risk to develop kidney stones, 37% compared with those in the highest quintile. Magnesium can inhibit calcium oxalate and calcium phosphate crystal growth and aggregation in vitro. The inhibitory activity of magnesium is positively related to urinary pH. However, magnesium oxide therapy could not show any therapeutic benefit in recurrent calcium stone formers. Pyrophosphates are found in hydroxyapatite and oxalate calculi in the urine as well. As pointed out above, pyrophosphates are natural inhibitors that block hydroxyapatite precipitation in vitro. In clinical studies, calcium stone formers had reduced urinary pyrophosphate concentrations. Urinary trefoil factor 1 is predominantly found in the stomach overlying the gastrointestinal mucosa. Recently, it could also be identified as a novel calcium oxalate crystal growth inhibitor in human urine by mass

spectrometry, with nearly the same inhibitory potential as nephrocalcin. [43]

Recently, the so-called crystal adhesion inhibitor, which is constitutively secreted by renal cells, could be identified. This 39 kDa protein blocked the adhesion of calcium oxalate crystals to the cell surface of epithelial cells. [43] _{A summary of actions of the described urinary calcium-}

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